IFN-γ enhances Poly(I:C)-induced necroptosis and immunogenic cell death via TLR3 upregulation in cholangiocarcinoma cells

Cholangiocarcinoma (CCA), a heterogeneous tumor arising in the bile ducts, is associated with unfavorable prognosis and high mortality rates due to limited effective treatment options. Though promising, immunotherapy is hindered by CCA's desmoplastic tumor microenvironment, known for its inflammatory and immunosuppressive characteristics, often termed a “cold tumor”. Previously, we reported that Poly(I:C), a TLR3 agonist, synergizes with a Smac mimetic, an IAP antagonist, to trigger necroptosis when caspase activity is blocked. In this study, we aimed to evaluate rational combination strategies to advance Poly(I:C)-based necroptosis immunotherapy in preclinical models. Transcriptomic analysis of public CCA patient datasets revealed that low expression of TLR3, accompanied by low IFNG or IRF1 expression, is significantly associated with poorer prognosis. These findings highlight the clinical relevance of the IFN-γ/IRF1/TLR3 axis and support the development of therapies targeting this pathway. We further found that IFN-γ treatment increases TLR3 expression. Importantly, we demonstrated that IFN-γ could enhance Poly(I:C)-induced necroptosis and immunogenic cell death (ICD) when IAPs and caspases are inhibited by Smac mimetic and the pan-caspase inhibitor Z-VAD-FMK in human CCA cell lines. CU-CPT 4a, a highly selective TLR3 inhibitor, partially reduced this cell death. This indicates that the effect is mediated in part through TLR3. The combined effects were shown to depend on RIPK1/RIPK3/MLKL-induced necroptosis. Additionally, the dying cells released HMGB1, a marker of ICD, and their conditioned medium promoted dendritic cell maturation (CD80, CD86, HLA-DR), which is critical for antigen presentation and T-cell priming. From a personalized medicine perspective, TLR3, IFNG, and IRF1 expression levels may serve as predictive biomarkers to guide patient selection for TLR3 agonist-based therapies. These findings could advance TLR3 ligand, Poly(I:C)-based necroptosis cancer immunotherapy, alone or with immune checkpoint inhibitors, for CCA and possibly other cancers.